Pain intensity and pressure pain threshold were evaluated up to 24 weeks after treatment. Adverse effects related to muscle contraction, masticatory performance, muscle thickness and mandibular bone volume were also assessed. Changes over time were compared within and between groups. The “nparLD” package and Wilcoxon signed-rank test were used to analyze the data. BoNT-A reduced pain intensity (p < 0.0001) and increased pressure pain threshold (p < 0.0001) for up to 24 weeks compared to the placebo. No differences were found between BoNT-A and OA at the last follow-up. A transient decline in masticatory performance (p < 0.05) and muscle contraction (p < 0.0001), and a decrease in muscle thickness (p < 0.05) and coronoid and condylar process bone volume (p < 0.05) were found as dose-related adverse effects of BoNT-A. Regardless of the dose, BoNT-A was as effective as OA on MFP. Notwithstanding, due to BoNT-A dose-related adverse effects, we suggest the use of low doses of BoNT-A in MFP patients that do not benefit from conservative treatments.This study assessed the safety and efficacy of three different doses of BoNT-A for persistent myofascial pain (MFP). One hundred female subjects were randomly assigned into five groups ( Persistent myofascial pain (MFP) is a common condition in dentistry with a prevalence of around 45% among subjects with temporomandibular disorders (TMD) . This diThe limited knowledge on the etiopathogenesis of MFP led to the proposal of numerous approaches to treat this condition ,4. ConseDespite the variety of published data about the management of persistent MFP with BoNT-A ,12,13,14Consequently, the development of new evidence-based and well-designed studies assessing the efficacy, possible AEs, and proper doses of BoNT-A, to establish it as a valid treatment for persistent MFP, became necessary. Therefore, we conducted a randomized controlled clinical trial to assess the efficacy and adverse effects of three different doses of BoNT-A compared with OA and placebo in subjects with persistent MFP. We tested the hypothesis that BoNT-A is more effective than saline solution (SS) and oral appliances for the treatment of persistent MFP.One hundred from a total of 540 volunteers were included into the study (mean age = 36.8 ± 5.6) . Charactp < 0.0001). In addition, no significant differences were found among BoNT-A groups. Compared to the placebo, subjective pain of BoNT-A groups was significantly lower (p < 0.0001) after 14 days and up to the end of the study; however, compared with OA, no statistical differences were found after 14 days and up to the end of the study (p > 0.05). A significant decrease of subjective pain A was fouWhen individual pain reduction (30%) was calculated in order to know if VAS improvement was clinically significant 20, 19, and 18 patients reported 30% pain reduction in BoNTA-L, M, and H groups respectively, compared to 20 and 13 patients in OA and SS groups respectively at the one month follow up. In the six-month follow up 20, 17, and 17 patients showed a 30% reduction in BoNTA-L, M, and H groups respectively, and 19 and 7 patients in OA and SS groups respectively .p < 0.01) after 14 and 21 days in masseter and anterior temporalis respectively and was maintained until the last evaluation. In addition, BoNT-A significantly improved PPT of both muscles after 21 days compared to placebo (p < 0.0001), sustaining this difference up to 180 days. Compared to OA, all BoNT-A groups presented significantly higher values on the 21 (p < 0.01) and 28 day (p < 0.001) evaluation for the anterior temporalis and in the 14 (p < 0.01), 21, 28 (p < 0.001) and 90 day (p < 0.01) for the masseter. No significant differences were found at the end of the experiment between BoNT-A and OA.The average PPT of the muscles is shown in p < 0.0001) compared with SS and OA. However, just BoNTA-L presented a significant recovery after 90 days and a total recovery of muscle activity at the last follow-up, presenting no significant differences to SS and OA in these evaluations’ points.Regarding electromyography (EMG) , BoNT-A p < 0.0001); however, a significant recovery was found after 90 days and maintained up to 180 days (p < 0.05). On the other hand, when BoNT-A and SS groups were compared no significant differences were found just for BoNTA-L in all evaluations (BoNT-A significantly decreased MP after 28 days (luations .p < 0.0001) in all evaluations. However, when BoNT-A groups were compared to SS, only BoNTA-L showed no significant differences at any evaluation.Ultrasound imaging (UI)  showed ap < 0.05). Comparison between groups showed no differences between BoNT-A and SS groups.Concerning cone beam computed tomography (CBCT) , the intRegardless of the dose, BoNT-A proved to be more effective than SS and at least as effective as OA to treat persistent MFP over 24 weeks. Notwithstanding, dose-dependent AEs were associated with higher doses of the toxin, but not found or easily overcome when a lower dose was employed. These results suggest that lower doses of BoNT-A are more appropriate when this treatment is contemplated for persistent MFP. We showed that even lower doses of BoNT-A can reduce subjective pain intensity (VAS) and increase PPT values over 24 weeks, indicating a potential analgesic effect independent of the accompanying dose-dependent motor activity . This anIn the present study, BoNT-A was significantly better than SS at reducing VAS and increasing PPT values throughout the six months. Likewise, in previous studies ,13, BoNTOAs are the most used therapy for persistent MFP due to their non-invasive and reversible characteristics . We founWithin the AEs, a reduction in electrical muscle activity was found after BoNT-A injections for up to the first month. Interestingly, only the BoNTA-L recovered after three months and returned to baseline values after six months. These results support a dose-duration effect for the toxin . In addiRegarding masticatory muscle thickness, a significant decrease in all evaluation periods was also identified among BoNT-A groups. Experimental studies demonstrated that muscle atrophy after BoNT-A injections responded to the decrease of fiber size , replaceThe musculoskeletal system maintains a fine regulation between muscles and bones, in which both tissues sustain a cross-talk process that may be affected by the neuroparalytic effect of the toxin. The decrease in the volume of mandible’s structures found in the BoNTA-H group supports this hypothesis. In vivo studies ,19 have As far as we know this is the largest study assessing the effectiveness and possible AEs of BoNT-A for persistent MFP. The design of our study, that included standardization of muscle thickness, positive and negative controls groups, three different doses of BoNT-A, and the assessment of its efficacy and safety, allow us to propose a safety margin for this treatment. Following this idea, we consider that in patients that do not get substantial pain relief from conservative treatments, lower doses of BoNT-A are the proper option when this treatment is contemplated for persistent MFP; since the possibility of developing AEs seems to be dose-dependent. Few limitations need to be considered. Longer follow-up periods should be evaluated in order to know the efficacy of a single injection of BoNT-L along time. The included sample was highly selected; therefore, our findings cannot be generalized to a broader population. Finally, even though we demonstrated that BoNT-A produces AEs, its clinical importance remains unclear. Future longitudinal studies should assess if AEs remain for longer periods of time and even if multiple applications of low doses of BoNT-A could develop AEs.It can be concluded that BoNT-A is at least as effective as OA for persistent MFP. However, due to dose-related AEs, conservative treatments  should be the first option for MFP. Notwithstanding, we suggest the usage of low doses of BoNT-A in patients that do not get substantial pain relief from conservative therapies. Further studies must be performed to refine BoNT-A’s indications.The present randomized, controlled clinical trial included 100 consecutive diagnosed subjects with persistent MFP according to the Portuguese version of the Research Diagnostic Criteria for Temporomandibular Disorders  at the Tn = 20): oral appliance , saline solution , BoNT-A low (BoNTA-L), BoNT-A medium (BoNTA-M), and BoNT-A high (BoNTA-H)  was operated by a technician not involved in any other procedures in the study. Subjects and investigators were masked to BoNT-A and SS assignments, while investigators assessing the outcomes were masked to all treatment assignments.Since BoNT-A effects are muscle-size dependent, subjects were submitted to ultrasonography of the masseter and anterior temporalis muscles to evaluate muscle thickness and equally divide them into above or below the median in each group . Subjects were then randomly assigned into one of the following treatment groups (BoNTA-H) . For thiCLS consisted of giving information about the anatomy and physiology of the stomatognathic system, the etiology and possible prognosis of persistent MFP, and given self-care strategies to control parafunctions. The same trained clinician performed CSL at the first appointment. The OA was a flat intraoral device covering all superior teeth, made of transparent thermo-polymerized acrylic resin. Subjects were instructed to use the OA during sleep throughout the study . AdjustmBoNT-A  was reconstituted using non-preserved sterile saline solution 0.9%. To blind the applications of BoNT-A and SS, each muscle received an injection containing 1 mL of the correspondent diluted BoNTA-L/M/H doses or SS by an investigator who was not involved in the dilution process. Doses of BoNT-A were based on previous reports ,12,13,14Subjects were evaluated eight times throughout the 6 months of study. Outcomes at each evaluation interval are presented in The VAS is a 100 mm horizontal line anchored by word descriptors at each end. The left end is labeled “no pain,” and the right “worst pain imaginable” . Partici2 circular point  used to exert pressure on the muscles. An increasing and constant pressure of 0.5 kg/cm2 was applied perpendicular to the surface of the skin [The PPT was assessed bilaterally for masseter and anterior temporalis muscles  in a relaxed position using a digital algometer 1 cmthe skin . SubjectTo record the electromyographic signal, ADS 1200 equipment  was used by a single calibrated operator (Kappa = 0.80). Bipolar electrodes were fixed in the most prominent part of each muscle after skin preparation and the reference electrode placed on the manubrium of the sternum of the volunteers . The LynThe multiple sieve method was used to assess MP. Cubical test foods of 5.6 mm were molded with condensation silicone  using a stainless-steel matrix. Then, each subject was asked to chew 17 cubes (3.4 g) 20 times in a habitual manner . 2 progression in a shaker  for 20 min. The particles retained in each sieve were weighed on a 0.001 g balance . The masticatory performance was calculated using a nonlinear regression (Rosin Rammler equation) [Afterward, subjects spat out the comminuted particles onto filter paper. After draining and disinfection, the filter with the particles was dried in an oven at 80 °C for 25 min. Each sample was sieved through a stack of up to 10 sieves with aperture sizes of 0.50 to 5.60 mm based on a √quation) .Thickness of both masseters and anterior temporalis muscles during maximum volunteer contraction (MVC) was calculated using real-time UI  by a single calibrated operator (Kappa = 0.86). Muscle thickness was measured directly on the instrument’s screen with an accuracy of 0.01 mm . The volume of the coronoid and condylar process of the mandible was assessed through CBCT . For this, CBCT multiplanar reconstructions were assessed with the semi-automatic mode of ITK-SNAP 3.4.0 segmentation software  . After sDue to limited funding sources we decided to assess MP, UI, and CBCT just in BoNT-A groups and in SS group.p < 0.001). Then, a two-way non-parametric test alternative was performed to analyze the interaction of time-points and treatment groups as simultaneous influence factors on variables. For non-parametric multiple comparison, in pairs, Wilcoxon signed-rank test with Bonferroni correction was used to show interaction between the time-points (paired measurements) (α = 0.05), and Mann–Whitney U with Bonferroni correction to groups (independent measurements) (α = 0.05) [The sample size was based on the average pain scores of a previous study . Power c = 0.05) . All var


The CGA-AuNPs obtained by the analysis of transmission electron microscope (TEM) images and ultraviolet–visible (UV–Vis) spectra were mainly spherical, and the average diameters were 18.94 ± 1.81, 30.42 ± 6.32, 37.86 ± 3.80 and 48.72 ± 6.47 nm, respectively. High-resolution transmission electron microscopy (HRTEM) and selected area electron diffraction (SAED) showed that these nanoparticles were polycrystalline gold structures. Both CGA-AuNPs and CGA have excellent antibacterial activity, and CGA-AuNPs with small particle size has a stronger antibacterial effect than the larger one. UV–Vis absorption spectrum data revealed that the synthesized CGA-AuNPs without adding other stabilizing agent were well maintained even after 26 days. This work provides a special idea to regulate the size of CGA-AuNPs with CGA by chemical synthesis, and the potent antibacterial activity of these CGA-AuNPs may be applied in the field of antibacterial in the future.This study proposes a strategy for the rapid and simple synthesis of gold nanoparticles (CGA-AuNPs) with different particle sizes using trisodium citrate (TSC) as the first reducing agent and chlorogenic acid (CGA) as the second reducing agent. And the antibacterial activity of CGA-AuNPs with different particle sizes  Various nanomaterials with singular functions have been developed and applied in energy, environment, biomedicine and other fields –3. AmongIt is worth noting that nano gold also has high antibacterial activity, especially against gram-negative bacteria , and it et al. successfully synthesized gold nanoparticles (CGA-AuNPs) using natural compound CGA as a reducing agent [et al. reported a photochemical synthesis method to regulate the size and shape of nanoparticles [In recent years, scientists have tried to find more green and natural products to synthesize nanomaterials –24, becang agent . Scaianoarticles . HoweverStaphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922). And the antibacterial effect of different sizes of CGA-AuNPs was viewed using an optical microscope. In addition, the absorbance of CGA-AuNPs with different sizes was measured by an ultraviolet spectrophotometer to evaluate the stability of CGA-AuNPs. The results showed that we have successfully synthesized CGA-AuNPs with different particle sizes and good stability, and CGA-AuNPs with different particle sizes also have the antibacterial effect. Under the same particle size, the antibacterial effect of CGA-AuNPs on S. aureus (ATCC 25923) was significantly better than E. coli (ATCC 25922).In this experiment, we chose CGA with antibacterial properties as one of the reducing agents for the synthesis of CGA-AuNPs with different particle sizes. The synthesis of different particle sizes of CGA-AuNPs regulated by CGA was characterized by ultraviolet–visible (UV–Vis) spectra, transmission electron microscrope (TEM), high-resolution transmission electron microscopy (HRTEM) and selected area electron diffraction (SAED). The antibacterial properties of CGA-AuNPs were verified by measuring the effect of CGA-AuNPs on the growth curves of 2.2.1.4 · 3H2O and CGA (1.18 × 10−6 mol l−1) in the reaction system was kept unchanged. With the increase of the concentration of TSC in the system, the position of the characteristic absorption peak of CGA-AuNPs was obviously blue-shifted (n(Au3+) : n (TSC) = 1 : 12. With the increase of the concentration of TSC, we found that the absorption value of CGA-AuNPs no longer increased and the characteristic absorption peak no longer shifted blue, indicating that almost all the gold ions in the system were reduced [Under the synthesis condition of 100°C, the concentration of HAuCl-shifted , indicat reduced .Figure 4 · 3H2O and TSC in the same concentration (molar ratios are 1 : 12) as above at 100°C. The CGA-AuNPs prepared with the increase of CGA concentration gradually shifted to the long-wave direction  in the wavelength range of 200–350 nm  were compared by one-way ANOVA. It was found that there was a significant difference  between each group, and the particle size of the nanoparticles has a tendency to become larger as the concentration of CGA increases (f), indicating that CGA can indeed regulate the particle sizes within this range, which was consistent with the UV–Vis absorption spectra of CGA-AuNPs  without CGA were used as the control group. As can be seen from figure 5b. Most of the CGA-AuNPs were stable within 8 h, and the absorption value of CGA-AuNPs of 48.72 ± 6.47 nm decreased slightly, which indicated that CGA-AuNPs had good stability in 50% LB medium at 37°C, and the stability of large particle size was slightly worse, which was consistent with the stability trend shown in figure 5a.The absorption values of CGA-AuNPs with different particle sizes at 520 nm in a 50% LB medium were also measured at 37°C within 12 h, and the results were shown in figure 52.5.E. coli (ATCC 25922) and S. aureus (ATCC 25923) are shown in figures 6f and 7f, it can be seen that the antibacterial effects of CGA-AuNPs (18.94 ± 1.81 nm) and CGA are basically the same, and the antibacterial effect was significantly reduced as the particle size become larger. ANOVA results showed that there were significant differences in the antibacterial effect between different particle sizes . This showed that particle size was one of the factors affecting the antibacterial effect. The possible reason is that small-sized CGA-AuNPs have a larger specific surface area and are, therefore, more likely to interact with cells. In addition, the level of bacteriostatic rate was also related to the CGA concentration. The bacteriostatic effect of different CGA concentrations was different . As shown in figure 7g and h, under the same conditions, the antibacterial effect of CGA-AuNPs on S. aureus (ATCC 25923) was higher than that on E. coli (ATCC 25922), which was consistent with the results of the control group. ANOVA showed that there were differences in the antibacterial effect between different bacteria . The reason for this phenomenon may be related to the surface reducing agent CGA. The presence of CGA may alter the interaction between nanoparticles and the two bacteria.The antibacterial activity of nanoparticles is influenced by many factors, in addition to the sizes of their own particles, and their shape, surfactant and type of reducing agent . The effS. aureus (ATCC 25923) and E. coli (ATCC 25922) treated with CGA or CGA-AuNPs were observed by optical microscopy. As shown in figure 8a and f, the two bacteria that were not added to CGA-AuNPs and CGA were intact, S. aureus (ATCC 25923) shown Gram-positive (blue-violet), and E. coli ATCC 25922 shown negative (red). After treatment with CGA-AuNPs for 8 h, the shape and size of the two bacteria changed, showing swelling, deformity and fragmentation, which may be caused by the accumulation of CGA-AuNPs inside the bacteria and the influx of water. Some of the S. aureus (ATCC 25923) was dyed red (figure 8c–e), indicating that the cell membrane was severely damaged. And the two bacteria treated by CGA (figure 8b and g) mainly showed swelling, but the morphology was basically intact, and the damage degree of the cell membrane was relatively small. This showed that the mechanism of CGA-AuNPs on bacteria was not exactly the same as CGA.In order to more clearly illustrate the antibacterial activity of CGA-AuNPs with different particle sizes, the morphological changes of Bacteriostatic experiments showed that the size of CGA-AuNPs is crucial for enhancing the antibacterial activity, so exploring the use of CGA to synthesizes smaller particle size CGA-AuNPs will be our future work. Meanwhile, CGA-AuNPs not only have good bacteriostasis, stability and biocompatibility but also have the advantages of nano gold, such as small size effect, optical effect, surface effect, low cytotoxicity and its modifiability, which make CGA-AuNPs have infinite potential in many fields waiting for our exploration.3.3.1.4 · 3H2O) were purchased from Shanghai Fine Chemical Materials Research Institute . TSC and CGA were purchased from Shenggong Biological Engineering Corporation . Strains of E. coli (ATCC 25922) and S. aureus (ATCC 25923) and Gram stain kit were purchased from Huankai Microbial Technology Corporation . All the chemicals were of analytical grade and were used without further purification. All the solutions were freshly prepared using ultra-pure water.Hydrochloroauric acid trihydrate . TEM images were obtained on a Tecnai 12 model . HRTEM and SAED were obtained on Tecnai G2 F30 S-TWIN .3.2.For the synthesis of the CGA-AuNPs, a hydrochloroauric acid trihydrate solution  was placed into a glass flask and heated to boil on a hot plate (set at 100°C) with stirring. To this solution, a solution of TSC  and CGA were added order and stirred on the hot plate for 12 min. Then, ultra-pure water (dilute to 100 ml) was added when the reaction mixture was naturally cooled. CGA-AuNPs of different particle sizes can be obtained by adding different volumes of CGA  according to the procedure described above.3.3.E. coli (ATCC 25922) and S. aureus (ATCC 25923) was observed by an optical microscope. E. coli (ATCC 25922) and S. aureus (ATCC 25923) were selected as representatives of Gram-negative bacteria and Gram-positive bacteria, respectively.The antibacterial activity of CGA-AuNPs with different particle sizes was measured by the growth inhibition curve method, and the antibacterial activity of these nanoparticles against E. coli (ATCC 25922) and S. aureus (ATCC 25923) were prepared by shaking culture at 37°C for 8 h in 150 r.p.m. incubator and inoculated in a 96-well plate. The initial density of bacteria was 1 × 107 CFU ml−1, and the final volume was 0.2 ml. Then, different concentrations of 1.8 ml CGA-AuNPs were added as the experimental group, and ultra-pure water was used instead of CGA-AuNPs as the control group. The 96-well plate was placed at 37°C and cultured in 150 r.p.m. incubator for 12 h. The OD600 value was measured every 1 h. The experiment was repeated three times, and three parallel samples were set at a time. ANOVA was used to examine the effects of different concentrations, sizes and strains on bacteriostatic effects.Firstly, the suspensions of The bacteria of the experimental group and the control group cultured for 8 h were stained with Gram and their morphology and quantity were observed under an oil microscope.4.In this study, antibacterial CGA-AuNPs ranged from 18.94 ± 1.81 to 48.72 ± 6.47 nm were successfully synthesized using CGA. CGA-AuNPs with small particle size have a stronger antibacterial effect. And they also have good stability.


In this case, the monodispersed alkali metal ions could realize highly effective activation with low activating agent dosage. Intensive material characterizations show that the alkali metal ions determine the pore structure and surface properties of as-prepared carbons. C-Cs prepared by Cs+ ion possesses a high Brunauer–Emmett–Teller specific surface area of 1,037 m2 g−1 with interconnected microporosity and sulfur doping. The specific capacitance of C-Cs can reach up to 270.9 F g−1 in a two-cell electrode measurement system, whereas C-Cs-based supercapacitors can deliver an energy density of 7.6 Wh kg−1, which is much larger than that of other samples due to its surface functionalities and well-interconnected porosities.A single alkali metal ion activation method was used to prepare sulfur-doped microporous carbons. A series of alkali metal ions such as Li Oxygen and nitrogen functionalities are believed to bring about pseudocapacitance by reacting with electrolyte, especially in an acid electrolyte. Numerous researches have been reported on the preparation of nitrogen- and oxygen-doped porous carbons with enhanced supercapacitive performance  specific surface area of 1,037 m2 g−1 with interconnected microporosity and sulfur doping. The specific capacitance of C-Cs can reach up to 270.9 F g−1 in a two-cell electrode measurement system, whereas C-Cs based supercapacitors can deliver a high energy density of 7.6 Wh kg−1 due to its surface functionalities and well-interconnected porosities.Considering the earlier mentioned views of porous carbon, constructing a microporous carbon with developed porous structure and heteroatom doping can contribute to high-performance electrode materials for supercapacitors. Herein, we prepared a series of sulfur-doped microporous carbons with the precursor of 3-hydroxythiophenol and formaldehyde. A series of alkali metal ions such as Li2O (volume ratio of 1:1); then, alkali hydroxide (8.7 mmol) and formaldehyde  were added into the mentioned transparent solution under magnetic stirring. The solution was solidified at 80°C for 24 h. The resulting hydrogel was cooled down to room temperature and immersed in acetone for 12 h to exchange the water with acetone. The exchange operation repeats four times, and the gel was removed from acetone and naturally dried to obtain xerogel before further carbonization. The xerogels are labeled as X-M, where M represents for alkali metal ion. The xerogel was carbonized in a tube furnace under Ar atmosphere at a flow rate of 50 ml min−1. The sample was heated at 200°C for 1 h with a heating ramp of 2°C min−1 and then carbonized at 700°C for 1.5 h with a heating ramp of 5°C min−1. The obtained carbon samples were washed with diluted HCl and deionized water to neutrality. The samples are labeled as C-M, where M stands for the alkali metal ion.In a typical synthesis, 3-hydroxythiophenol  was dissolved in 8-ml ethanol/H2 gases with super high purity (99.999%) were used for the physisorption measurements. The BET equation was used to calculate the apparent surface area from N2 adsorption data. For advanced porosity analysis, pore size distributions, cumulative surface areas, and cumulative pore volumes were determined by using quenched solid-state density functional theory (QSDFT) method considering sorption of nitrogen at 77 K in carbon as a model adsorbant and slit-like pores as a pore model. The Quantachrome Autosorb ASiQwin 2.0 software supplied the implemented QSDFT model.The morphology of the samples was observed using a scanning electron microscope  and transmission electron microscopy . The crystal structure of the C-Ms was determined by X-ray diffraction  using Cu Kα radiation (λ = 0.15418 nm) and Raman spectroscopy  using a 632.8-nm wavelength laser. The surface properties of C-Ms were characterized by X-ray energy dispersive spectroscopy  and X-ray photoelectron spectroscopy . Nitrogen adsorption–desorption isotherms were measured at liquid nitrogen temperature (77 K) using a surface area and porosity analyzer . The carbon samples were degassed under a turbomolecular vacuum at 300°C for 6 h before sorption measurements. NCm = 4It/Vm, where Cm, I, t, V, and m are the specific capacitance, discharge current, discharge time, potential window, and the total mass of active materials in the cell, respectively. The energy density (E) and power density (P) of the cell could be calculated according to the equations: E = CmV2/(8 × 3.6), P = 2E/t, where Cm, t, and V are the specific capacitance, discharge time, and potential window in the cell, respectively.Working electrodes were made by pressing the electrode materials onto nickel foam, in which the electrode materials contained 5 wt% polytetrafluoroethylene as a binder. The mass of active materials in each working electrode is about 2 mg. To ensure that the active materials are thoroughly wetted with the electrolytes of 6-M potassium hydroxide, the working electrode was vacuum-impregnated with the electrolyte before electrochemical tests. The electrochemical capacitive performances of samples were studied on a CHI660E electrochemical workstation. All electrochemical measurements, including cyclic voltammetry (CV) and galvanostatic charge–discharge, were performed in a two-electrode system using two electrodes. A potential window of 0–0.9 V (6-M KOH electrolyte) was applied to the electrochemical measurements. The specific capacitance was calculated from the discharge curves according to the equation SEM images of X-Na  show tha2 carbon domains  and reaches a plateau afterward, indicating a micropores-dominated pore-structure for C-K and C-Cs. Isotherms for C-Li and C-Na show the same characteristic in the low relative pressure region (P/P0 < 0.45), suggesting the presence of micropores in these samples. However, the presence of a hysteresis loop in C-Li and the sharp increase of adsorbed quantities in the high relative pressure region in both samples suggest that there are numerous mesopores and macropores in C-Li and C-Na. The adsorbed quantity of plateau (P/P0 < 0.4) for each sample obeys the following order: C-Cs > C-K > C-Na > C-Li, suggesting that micropore content obeys the same order as the adsorbed quantity in this region is contributed from micropores. The BET surface areas and total pore volumes  show that all of the discharge branches for C-Ms are linear shape, suggesting the EDL capacitance-dominated energy storage mechanism for all samples. The charge branches for C-Na, C-K, and C-Cs present a non-linear characteristic, especially in the potential range of 0.7–0.9 V due to the insertion of ions into the inner pores, corresponding well with the CVs in −1  and much higher than that of C-Na (27.9 %) and C-Li (16.7 %). Combining the results of structural analysis, such a good capacitance retention ratio for C-Cs is possibly due to the high surface area, interconnected pore structure, and heteroatom doping in C-Cs and C-K. The high surface area and interconnected pores can provide a high interface for electrolyte, whereas the heteroatom doping can increase the wettability of carbon surface to an electrolyte, thus accommodating more electrolytes on the interface to generate high capacitance even under high current densities.The charge branch for C-Cs shows increased response current densities within the potential range of 0.7–0.9 V, which is probably due to the insertion of ions into the inner pores of C-Cs. As measured by nitrogen sorption analysis, such variation is probably due to the presence of numerous micropores in C-Cs. However, the CVs for all samples at a scan rate of 500 mV s samples . The gal samples  at low cVs in −1 . The speVs in −1 . As tabuBET values for C-Li and C-Na are 40.92 and 38.59 μF cm−2, respectively, much higher than that of C-K and C-Cs. The low areal capacitance for C-K and C-Cs can be attributed to the lower content of heteroatom functionalities, which brings about pseudocapacitance for C-Ms. This result could coincide well with the CV analysis, suggesting the presence of significant pseudocapacitance generated by heteroatoms in C-Na and C-Li. As for C-K and C-Cs, their areal capacitances are higher than those reported of KAC and CsAC with a similar preparation method. Such higher areal capacitance could be attributed to the extra sulfur doping in C-K and C-Cs. Moreover, the areal capacitance of C-Cs (15.76 μF cm−2) at high current density (20 A g−1) is still higher than that of CsAC (12.35 μF cm−2), as CsAC has a higher carbon content, which probably contributes to a better conductivity in comparison with C-Cs. The lower areal capacitance for CsAC is attributed to its absence of sulfur functionalities, which can improve the hydrophilicity for porous carbon and accommodate more electrolyte ions even at high current densities. With high specific surface area, well-interconnected porous structure, and oxygen and sulfur doping for C-Cs, supercapacitor cells assembled by C-Cs can deliver an energy density of 7.6 Wh kg−1 (The impact of heteroatom on the capacitive property is also investigated in our work. The areal capacitances calculated from BET specific surface area are tabulated in  Wh kg−1 , which i−1. Our research proposes an effective strategy on the design of porous and surface structure for carbon-based energy storage materials.Sulfur-doped porous carbon activated by alkali metal ions is prepared through an alkali metal ion activation method in this paper. Such a preparation method with alkali metal ions uniformly anchored in the sulfur-containing resin can realize the high-effective activation by generating interconnected porosity and concentrated pore size distribution. Porous carbon shows an increased surface area as the increasing of adopted metal ions' radius. On the other hand, luxuriant surface oxygen/sulfur functionalities inherited from carbon precursor in porous carbon can effectively improve the hydrophilicity of carbon surface to electrolyte and bring about extra pseudocapacitance. In this case, supercapacitor assembled with C-Cs can deliver an energy density of 7.6 Wh kgAll datasets presented in this study are included in the article.CW did all the work containing experiments, data and manuscript. The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Conus purpurascens, which is essential for quick prey’s excitotoxic immobilization. Binding of one κ-PVIIA to Shaker K-channels occludes the K+-conduction pore without additional conformational effects. Because this 27-residue toxin is +4-charged at neutral pH, we asked if electrostatic interactions play a role in binding. With Voltage-Clamp electrophysiology, we tested how ionic strength (IS) affects κ-PVIIA blockade to Shaker. When IS varied from ~0.06 to ~0.16 M, the dissociation constant for open and closed channels increased by ~5- and ~16-fold, respectively. While the association rates decreased equally, by ~4-fold, in open and closed channels, the dissociation rates increased 4–5-fold in closed channels but was IS-insensitive in open channels. To explain this differential IS-dependency, we propose that the bound κ-PVIIA wobbles, so that in open channels the intracellular environment, via ion-conduction pore, buffers the imposed IS-changes in the toxin-channel interface. A Brønsted-Bjerrum analysis on the rates predicts that if, instead of fish, the snail preyed on organisms with seawater-like lymph ionic composition, a severely harmless toxin, with >100-fold diminished affinity, would result. Thus, considerations of the native ionic environment are essential for conotoxins evaluation as pharmacological leads.κ-Conotoxin-PVIIA (κ-PVIIA) is a potassium-channel blocking peptide from the venom of the fish-hunting snail,  Conus that bind to a wide variety of transporters, ion channels, G protein-coupled receptors and enzymes, providing a rich source of molecular templates and therapeutic leads for drug developing [Conus purpurascens, a piscivorous marine snail, that possibly is partially responsible for the excitotoxic shock in fish [−1μM−1, a value common among α-KTx toxins and in the range of diffusion limited protein associations [Conotoxins form a family of peptides produced by marine predatory snails of genus veloping . In fishveloping . κ-conot in fish . The tox in fish ,4,5. Add in fish ,7. Thus,ciations ,9. Such ciations .As other animal toxins of the same family, κ-PVIIA is endowed with an excess of positive charges at its surface ,11. The Shaker K-channels. We found that the association rate is very sensitive to the ionic strength, which is a result consistent with our hypothesis that the protein-protein recognition is mediated by through-space electrostatic interactions. Interestingly, the dissociation rate is also sensitive to the ionic strength in closed channels, but in open channels is ionic strength insensitive. We suggest that this lack of sensitivity in open channels is due to the fact that the toxin wobbles in the bound state [In this work we study the effects of ionic strength in the kinetics of κ-PVIIA blockade on nd state .Shaker K-channels in Xenopus oocytes was detected as positive (outward) currents in response to positive-going voltage pulses between −80 and +50 mV from a holding potential of −90 mV . In the presence of κ-PVIIA (right traces), the activation kinetics appears delayed and the currents at the end of the voltage pulse slightly diminished in relation to their controls. As the ionic strength increases, the toxin induced inhibition gets weaker. Unfortunately, we had to limit the ionic strength excursion up to 0.16 M because higher ion concentrations induce osmotic stress that damages the oocytes and the quality of the recordings.In normal amphibian plasma ionic strength, heterologous expression of voltage activated f −90 mV . These vWe have shown before that the apparent effect on the kinetics is due to the voltage dependence of the toxin/channel binding equilibrium ,5,13. ThTx/iCon; filled circles in All quotient traces start from approximately the same level of inhibition at the beginning of the voltage pulse . Thus, as expected, increased ionic strength decrease κ-PVIIA affinity for potassium channels, but interestingly, binding to a closed channel seems ~4-fold more sensitive to the ionic strength than it is to open channels. This difference could reveal a different electrostatic environment sensed by the toxin when interacting with open or closed channels. In order to address the mechanistic basis of such differential effects we determined the effect of ionic strength on the rate constants for toxin binding and unbinding in open and closed channels.As the ionic strength increases, the inhibition of resting and open channels decreases. But the ionic strength effect on the inhibition at resting seems to be much more intense than that of the open state. Thus, for example, the resting fractional current rises from ~0.15 at 50-NaTx/iCon) in on and koff, respectively, according to the following equation system:on and koff for each voltage. +, 100-Na+ and 150-Na+. As it happens with scorpion toxins having diffusion limited and electrostatically aided association rate to potassium channels [on is voltage independent, showing most of the ionic strength dependency, being at 150-Na+ 20–25% of that calculated for 50-Na+. On the other hand, the voltage dependent dissociation rate, koff increases marginally at 150-Na+.We and others have previously shown that a 1:1 stoichiometry describes adequately κ-PVIIA binding equilibrium ,5,13,14.channels ,15,16, kon and koff for the binding to the closed state. on changes about the same ~5 fold for both, open and closed channels (black circles in Open and Closed panels), with the excursion from 50-Na+ to 150-Na+, the koff’s had opposite dependency on the ionic strength.To obtain the kinetic constants of κ-PVIIA binding at resting, we used a two-pulse protocol devised by Terlau et al.  to test A = +4e, according to the predicted peptide charge at neutral pH [B, and the rate constant at zero IS, k0. For the association rates to open and to closed channels, ZB was ~−2.5 e, suggesting that, regardless of the structural details, open and closed channels display similar electrostatic landscapes for the approaching κ-PVIIA. In contrast, unbinding rates from open and closed channels show a dramatic difference in their dependence on the ionic strength. On one hand, in open channels the IS-rise slightly slows unbinding, but in closed channels it conspicuously destabilizes the complex. Therefore, the fit parameter accounting for this difference, ZB, changes form −0.5 in open channels to ~+2.2 in closed ones, indicating different electrostatic surroundings for the bound toxin. We propose that the open channels reduced ionic strength sensitivity is suggestive that κ-PVIIA, as CTX, wobbles in the potassium channel mouth [A decreasing association rate with the ionic strength is expected if the toxin and the channel are oppositely charged, thus, the attractive force between them is weakened by the interposed ionic cloud. As a simple quantitative analytical approach we used the Brønsted-Bjerrum equation to fit the kinetic data as function of the ionic strength. This equation predicts that the rate has an exponential dependency on the product of the charges and √IS (see Equation 5)). To do the fits, we fixed the κ-PVIIA charge, Zutral pH . Thus, o). To do We will discuss two ideas: (A) Unbinding of κ-PVIIA from open channels is less sensitive to changes in ionic strength than the unbinding from the closed ones. We suggest that because of toxin wobbling, the external mouth of the blocked pore equilibrates with the internal (unchanged) solution in the open channel, buffering the effect of the ionic strength of the external medium. In the closed channel such equilibration does not occur because the pore communication with the internal solution becomes interrupted by the closed channel activation gate . (B) Theon for open and closed channels exhibits similar ~5-fold decrease from 50 to 150 mM of external NaCl (50-Na+ to 150-Na+), which, according the Brønsted-Bjerrum equation (Equation (5)), is expected for a bimolecular reaction between oppositely point-charged reactants having a charge-product, ZA × ZB ~−10eo2 , whose lymph has similar ionic composition as sea water ,28. InstXenopus laevis anesthetized by immersion in ice. Ovarian lobes were surgically removed and collected in ND96 solution (in mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, pH 7.6 . Collagenase  was used at a concentration of 0.9–1.5 mg/mL for digestion of tissue. After washing the enzyme, the oocytes were manually defolliculated in a nominally Ca2+-free ND96 solution.Methods and reagent in general and κ-PVIIA are as in . BrieflyShaker K channel was expressed in Xenopus oocytes with in vitro transcribed cRNA injection (ranging from 50 to 200 pg per oocyte). The oocytes were incubated at 18 °C in ND96 supplemented with sodium pyruvate (2.5 mM) and BSA (0.04%) until subsequent recording. Macroscopic potassium currents were recorded with the two electrode voltage clamp techniques (TEVC) under continuous perfusion with a buffered saline containing (in mM): 96 NaCl, 2 KCl, 1 MgCl2, 0.3 CaCl2, 10 HEPES, and 25 μg/mL BSA, pH 7.6 (100-Na+). We used 0.3–1 MΩ microelectrodes, filled with 3M KCl, 5 mM EGTA and 10 mM HEPES (pH 7.0). The ionic strength was manipulated by adding extra 50 mM NaCl to the solution (150-Na+) or by replacing 50 NaCl with 100 mM mannitol in the solution (50-Na+). Recorded outward currents were evoked by voltage steps from –80 mV to +50 mV, from a holding potential of −90 mV (200 ms duration). Aliquots of κ-PVIIA were added into the bath solution immediately prior to perform the recordings. Calculations of the dissociation and association rate constants, koff and kon, respectively, of κ-PVIIA blockade on open channels, were made from equation system (1) A concentration of 100 nM of toxin in the bath was used to measure both rate constants in open channels. The remaining fractional current at equilibrium (iTx/icon) was used to obtain the dissociation constant, KD, with:on was determined with:The inactivation-removed 0 is the kinetic constant at zero ionic strength, ZA and ZB are the ion´s electronic charge and IS, the ionic strength in M1/2. For all fits ZA was fixed to +4 according to the theoretical κ-PVIIA charge [0 and ZB (the channel charge) were left as fitting variables. We calculated IS according to the following formula:i is the concentration (in mol/L) of ion i and Zi, its valence. All fits were made in Origin 6 (originlabs.com) with the Levenberg-Marquardt method.To predict the toxin binding equilibrium behavior in fish or mollusk plasma ionic condition we used the standard Brønsted-Bjerrum equation that evaluates the kinetic constants of a pseudo-first or second order reaction between ions .(5)k=koA charge  and only


Microfluidic devices enable the precise operation of liquid samples in small volumes. This motivates why microfluidic devices have been applied to point-of-care (PoC) liquid biopsy. Among PoC liquid biopsy studies, some report diagnostic reagents being freeze-dried in such microfluidic devices. This type of PoC microfluidic device has distinct advantages, such as simplicity of the procedures, compared with other PoC devices using liquid-type diagnostic reagents. Despite the attractive characteristic, only diagnostic reagents based on the cloned enzyme donor immunoassay (CEDIA) have been freeze-dried in the microfluidic device. However, development of the PoC device based on the CEDIA method is time-consuming and labor-intensive. Here, we employed a molecule-responsive protein synthesis system as the diagnostic reagent to be freeze-dried in the microfluidic device. Such molecule-responsive protein synthesis has been well investigated in the field of molecular biology. Therefore, using the accumulated information, PoC devices can be efficiently developed. Thus, we developed a microfluidic device with an integrated freeze-dried molecule-responsive protein synthesis system. Using the developed device, we detected two types of bio-functional molecules  by injecting 1 µL of sample solution containing these molecules. We showed that the developed device is applicable for small-volume biosensing. Microfluidic devices make it possible to analyze sample solutions even in small volumes. In fact, the sample volume required to analyze with microfluidic devices is smaller than that of a general plate reader ~5 µL). This advantage is useful to reduce invasiveness in liquid biopsy using blood, urine, saliva, and tears. Therefore, microfluidic devices have been applied as point-of-care (PoC) diagnostic devices ,2,3,4. R µL. ThisThe molecule-responsive protein expression system consists of a cell-free protein synthesis (CFPS) system and DNA encoding a reporter protein under a molecule-responsive promoter. CFPS systems generally contain RNA polymerase, ribosome, nucleotides, amino acids, adenosine triphosphate regeneration system, and salts ,9. The sThe microfluidic device was fabricated using standard soft-lithography . An SU8 N-acyl-homoserine-lactone (AHL) or Hg2+. The procedures to construct these DNAs are described below.In this study, we constructed two types of molecule-responsive DNAs. These DNAs were activated to synthesize GFP in response to http://n2t.net/addgene:40019; RRID: Addgene_40019) was a gift from Vincent Noireaux of the University of Minnesota  [Vibrio fischeri under its native promoter and deGFP under the LuxI promoter. The LuxI promoter is activated by the AHL-luxR complex; therefore, deGFP is expressed in response to AHL.AHL-responsive DNA was constructed from pRMC004, encoding the LuxI promoter, and pBEST-OR2-OR1-Pr-UTR1-deGFP-T500 encoding the green fluorescent protein (GFP). pRMC004 was a gift from Robert Cooper of the University of California, San Diego, CA, USA). pBEST-OR2-OR1-Pr-UTR1-deGFP-T500  . The Luxmer) and deGFP gene. The Pmer part also encoded the MerR protein under a constitutive promoter. Pmer was activated by the MerR-Hg2+ complex and repressed by the MerR dimer [mer. Therefore, GFP expression occurs when Hg2+ ions are present; conversely, no GFP expression occurs when Hg2+ ions are not present. The DNA sequence of Pmer was obtained from the literature [mer was synthesized by a DNA-synthesizing company . The DNA sequence, except for the Pmer, was subcloned from pBEST-OR2-OR1-Pr-UTR1-deGFP-T500. These two DNAs were connected using NEBuilder HiFi DNA Assembly Master Mix. The constructed circular DNA was transformed into DH5α chemically-competent cells (Takara). The plasmid DNA  was amplified by culturing the cells and purified using the GenElute HP Plasmid Maxiprep Kit.Mercury-responsive DNA consisted of a mercury-responsible promoter  when necessary.The CFPS system was purchased from Promega . We used the CFPS system according the manufacturer’s instructions. To sense AHL, pAHL-deGFP was added to the CFPS system; similarly, to sense mercury ions, pHg-deGFP was added.The CFPS solution, containing DNA , was introduced into the microchambers of the microfluidic device and freeze-dried. First, the microfluidic device was placed in a vacuum chamber for 20 min. Next, 1 µL of the CFPS system, containing the designated concentration of DNA, was injected from the inlet using a pipette. At this time, the CFPS solution was absorbed into the microchambers . After aTo test the aqueous sample, the microfluidic device was first placed in the vacuum chamber for 20 min. Next, 1 µL of the sample solution was injected from the inlet using a pipette. After injection, the sample solution was absorbed into the microchambers to rehydrate the freeze-dried CFPS system. The remaining air bubbles in the microchambers were absorbed into the PDMS walls to disappear typically within 2 min a–c. Fina0) as (F−F0)/F0 (denoted as ΔF/F0).Fluorescent intensities of the CFPS solutions were monitored using a fluorescent microplate reader  with a fluorescent filter unit for GFP. 9.5 µL of the sample solutions were applied to each well of a 384-well plate (Merck). During the measurement, the microplate was kept at 37 °C so that the CFPS system synthesized proteins at high speed. The obtained fluorescence intensities (F) were normalized by the initial intensities (F0) as (F−F0)/F0 (denoted as ΔF/F0).Fluorescence intensities of the microchambers were monitored using an inverted fluorescent microscope  with a fluorescent filter unit for GFP  and a monochromic CCD camera . Before monitoring, the inlet and outlet were sealed with vacuum grease to prevent dehydration. The microfluidic device was then placed in a Petri dish with water droplets to maintain the humidity. The microfluidic device in the Petri dish was maintained at 37 °C using a heating plate . The obtained fluorescent images were analyzed using image processing software . The fluorescence intensities in the microchambers (F) were normalized by the initial intensities (Fconst) . The DNA encoded deGFP under a constitutive promoter (Pconst) a. We rehconst) c. This rconst) and deGFP gene under the LuxI promoter (PluxI)  and 0.5 mM EDTA with or without 10 μM AHL. After rehydration with 1 μL of the aqueous solution containing AHL, the average fluorescence intensity from the microchambers gradually increased  a. The AH complex . The Luxncreased b. MeanwhWe finally examined mercury ion sensing. Drinking water and sources of drinking water  are supposed to be quality checked periodically. The inspection items include contaminants that can be toxic to human health, such as mercury, cadmium, arsenic, lead, chromium, polychlorinated biphenyl, cyanide, and benzene ,23. Amon2+ ions. pHg-deGFP was expected to synthesize GFP when the Hg2+ concentration was higher than the threshold, as shown in First, we confirmed that the constructed mercury sensing DNA (pHg-deGFP) could detect Hg2+ ions. 2+ at various concentrations of pHg-deGFP. This indicates that as the pHg-deGFP concentration increased, GFP expression levels also increased in the presence of 200 nM of Hg2+ ions. It also shows that even if the pHg-deGFP concentration was high, GFP expression levels did not change dramatically in the absence of Hg2+ ions. Therefore, higher concentrations (≥ 100 ng/µL) of pHg-deGFP are suitable for the detection of Hg2+ ions, in fact 200 ng/µL was used for subsequent experiments.Then, we investigated which pHg-deGFP concentration was suitable for the detection of Hg2+. The graph shows that 20 nM of Hg2+ was detected without overlapping error bars against 0 nM. However, 5 nM of Hg2+ was not detectable because the error bars of 5 and 0 nM overlapped. From this result, the detection limit was found to be 20 nM. This value is lower than the WHO standard (~30 nM) [Next, we investigated the detection limit of the mercury sensing system. (~30 nM) .2+ (Milli-Q water with/without 200 nM of HgCl2). 2+. From this result, we conclude that the developed microfluidic mercury sensor could detect Hg2+ ions.We next confirmed that mercury ions could be detected in the developed microfluidic device with the freeze-dried CFPS system and the freeze-dried pHg-deGFP DNA. We freeze-dried the CFPS solution containing 200 ng/µL of pHg-deGFP in the microchambers of the microfluidic device. After freeze-drying, the freeze-dried CFPS system with pHg-deGFP in the microchambers was rehydrated with water with/without 200 nM Hg2 to obtain 50 nM of Hg2+ in one of the water samples. The water samples were directly applied to the microfluidic device without any treatment. p-value was calculated to be ~0.04 using Welch’s t-test. This result indicates that 50 nM of Hg2+ in the river water sample is detected after 6.5 h. From this result, we conclude that the developed device can be applied for water quality testing/monitoring. The variance of fluorescent intensities in 2+ concentration in this sample solution (50 nM) is close to the dissociation constant between MerR and Hg2+ . Around2+ sensing, using the developed device integrated with the freeze-dried CFPS system. For sensing AHL and Hg2+, the AHL-responsive promoter and the Hg2+-responsive promoter were employed. By having these promoters before the GFP gene in the DNA sample, DNA activation by AHL or Hg2+ was enabled and could be validated through the GFP signal. Using the developed device with these DNA samples, we detected 10 µM of AHL and 200 nM of Hg2+. Notably, the simple operation via the injection of an aqueous sample solution as low as 1 µL enables biosensing. We also demonstrated water quality testing with this microfluidic device by injecting 1 µL of sample obtained from the river into the device: We could detect 50 nM of Hg2+ that was artificially added to the river water.This study aimed to develop a biosensing device that can be used via simple operation with a small volume of samples. To achieve this concept, we developed a microfluidic device that has a freeze-dried CFPS system in its microchambers. The device was fabricated by freeze-drying the CFPS solution in the microchambers. To prove this concept, we first validated that the freeze-dried CFPS system can express proteins from freeze-dried DNA after rehydration with 1 µL of the aqueous solution injected from the inlet. Next, we demonstrated two types of biosensing, AHL and HgAs shown in this study, the microfluidic device developed with the freeze-dried CFPS system enables small-volume biosensing. In addition, by changing the promoter region in the DNA, sensing devices responsive to other molecules can be developed. Therefore, this technology will be useful for on-site biosensing applications such as PoC diagnostics and water quality testing.


Structure delineation is a necessary, yet time-consuming manual procedure in radiotherapy. Recently, convolutional neural networks have been proposed to speed-up and automatise this procedure, obtaining promising results. With the advent of magnetic resonance imaging (MRI)-guided radiotherapy, MR-based segmentation is becoming increasingly relevant. However, the majority of the studies investigated automatic contouring based on computed tomography (CT).In this study, we investigate the feasibility of clinical use of deep learning-based automatic OARs delineation on MRI.95), and mean distances were calculated against clinical delineations. For eight patients, an expert RTT scored the quality of the contouring for all the three methods. A choice among the three approaches was made, and the chosen approach was retrained on 97 patients and implemented for automatic use in the clinical workflow. For the successive 53 patients, Dice, HD95 and mean distances were calculated against the clinically used delineations.We included 150 patients diagnosed with prostate cancer who underwent MR-only radiotherapy. A three-dimensional (3D) T1-weighted dual spoiled gradient-recalled echo sequence was acquired with 3T MRI for the generation of the synthetic-CT. The first 48 patients were included in a feasibility study training two 3D convolutional networks called DeepMedic and dense V-net (dV-net) to segment bladder, rectum and femurs. A research version of an atlas-based software was considered for comparison. Dice similarity coefficient, 95% Hausdorff distances (HDDeepMedic, dV-net and the atlas-based software generated contours in 60 s, 4 s and 10-15 min, respectively. Performances were higher for both the networks compared to the atlas-based software. The qualitative analysis demonstrated that delineation from DeepMedic required fewer adaptations, followed by dV-net and the atlas-based software. DeepMedic was clinically implemented. After retraining DeepMedic and testing on the successive patients, the performances slightly improved.High conformality for OARs delineation was achieved with two in-house trained networks, obtaining a significant speed-up of the delineation procedure. Comparison of different approaches has been performed leading to the succesful adoption of one of the neural networks, DeepMedic, in the clinical workflow. DeepMedic maintained in a clinical setting the accuracy obtained in the feasibility study. Structure delineation is a necessary, yet time-consuming manual procedure in radiotherapy. Consistent and accurate delineation of organs-at-risk (OARs) and target structures for prostate patients is vital when performing dose escalation and treating patients with highly conformal plans . TraditiThe manual segmentation of anatomical structures is a time-consuming process . BesidesTo automatically perform delineations of target and OARs for patients affected by prostate cancer, various methods have been developed over the past years. For example, three-dimensional (3D) deformable model surface , organ-bRecently, deep learning has been proposed to speed-up and automatise automatic segmentation obtaining promising results , 21, 22.Many studies focused on target delineations  reachingIn this study, we aim at investigating the feasibility of convolutional neural network-based automatic OARs delineation on MRI. A preliminary retrospective study was conducted to select a suitable network architecture and prepare for clinical implementation. After having chosen the most suitable convolutional network and performing clinical implementation, performances of automatic deep learning-based OARs delineation from our clinic are presented.Patients diagnosed with intermediate and high-risk prostate cancer undergoing MR-only radiotherapy  in the pⓇ for Image Registration 128, Beekley Medical, USA) were used to identify the set-up location on MRI. MR images were acquired using anterior and posterior phased array coils . Two in-house-built bridges supported the anterior coil to avoid skin contour deformation.For all patients, 3T MRI  was acquired after requesting the patients to empty their bladder and drink 200-300 ml of water one hour before the acquisition. Patients were positioned on a vendor-provided flat table using a knee support cushion . Patients were tattooed at the MRI with the aid of a laser system  to facilitate treatment positioning. Also, MR-visible markers (PinPoint OARs were contoured on Dixon images  obtainedThe first 48 patients (treated until January 2019) were included in a feasibility study training two state-of-the-art 3D convolutional networks called DeepMedic  and densThe performance of the networks was compared against a research version of commercial software based on multi-atlases and deformable registration and against the clinically used delineations “” sectionThis preliminary study enabled us to choose among the three automatic methods. The preferred approach was retrained on 97 patients that were imaged and treated until August 2019; it was implemented for automatic use in the clinical workflow. The performances of the implemented model were reported on the 53 successive consecutively treated patients. A schematic overview of the study design is presented in Fig. σ), and then a fixed value of 1 was subtracted.Three-dimensional network architectures were chosen to investigate performance differences considering as perceptive field the whole volumes or smaller patches. In particular, DeepMedic  was the After training and inference of the networks, the delineations were post-processed generating four binary volumes. Morphological operations of closure and hole filling by one voxel were applied. The largest 3D connected region was selected for each delineated structure. These operations were performed to remove possible small-sized delineations that may have been found by the networks.3, 513, 173 voxels were employed with each pathway consisting of 11-layers. A fully connected network (FCN) was used for combining the pathways and post-processing, as presented by Kamnitsas et al. [The DeepMedic  implemens et al. . Note thI) by the following: I′=(I+s)∗m, where s and m where Gaussian distributed with μ=0, 1 and σ=0.05, 0.01, respectively. For training, DeepMedic made use of about 9 GB of GPU memory.The training configuration was kept as the original, with learning rate = 0.001, Adam optimiser with momentum = 0.6, epochs = 35, batch size = 10 and entclass1pt{minimaThe dV-net implementation provided in NiftyNet was employedThe training configuration was kept as the original, with learning rate= 0.001, Adam optimiser with momentum = 0.6, batch size = 6, The first 48 patients treated between June 2018 and August 2019 were included in a preliminary study to compare the performance of the two networks and atlas-based approach to the delineation used during clinical treatment planning.The advanced medical imaging registration engine  was the software considered; ADMIRE is based on multi-atlases , 44 and 95) [https://github.com/deepmind/surface-distance. In particular, violin plots [σ, 95% percentile and the probability distribution were obtained for the three metrics. Also, Wilcoxon signed-rank tests were conducted among the three evaluation metrics with a confidence interval of 0.05.Performances of the three automatic approaches were evaluated in terms of (volumetric) dice similarity coefficients (DSC), 95% boundary Hausdorff distances (HD95) , mean suin plots  represenFor a subset of 8 patients, an RTT with five years of experience in contouring scored the quality of the delineations for all three methods. The delineations were classified from zero to three, which corresponds to clinically acceptable, small modifications, large modifications, or clinically unacceptable contours. In total, the RTT scored 96 delineations. The percentage of each score over all the contours was reported for the three methods and visualised in a pie chart. Also, the most challenging structures (structures with an average score ≥2) were reported for each method.95 and MSD for the 53 consecutive patients undergoing MR-only radiotherapy from August 2019 to January 2020. The delineations adopted for clinical use, i.e. delineated by RTTs and approved or re-adjusted by a radiation oncologist, were considered as reference. Also, surface dice similarity coefficient (SDSC) [After a choice was made among the three automatic approaches, the best performing network was retrained for the first 97 patients that were included up to August 2019. The hyperparameters were identical to the preliminary study. The network was implemented for clinical use complying with the medical device regulation (MDR 2017/745)t (SDSC)  was calct (SDSC) . BesidesThe inference time of the network was about 60 s for DeepMedic and approximately 4 s for dV-net using the full resolution images of 328x328x120 voxels on GPU. ADMIRE generated contours in approximately 14 min on GPU.95 and the MSD. One can observe that performances were higher for both the networks compared to ADMIRE. For the bladder, no significant differences were observed between the networks, but significant differences were observed between the networks and ADMIRE. For the rectum, no significant differences were observed among the three automatic methods. When considering the femurs, DeepMedic outperformed both dV-net and ADMIRE. For example, for the right femur, the mean (±σ) HD95 was 2.2 ±1.4, 2.5 ±1.8 and 3.2 ±1.4 mm for DeepMedic, dV-net and ADMIRE, respectively.Figure The qualitative scoring by an RTT expert Fig.  demonstrOn the basis of the preliminary analysis, we decided to implement DeepMedic for our clinic. Clinical implementation was performed in August 2019.95 and MSD after retraining the network on a more extensive set was ameliorated by 0.01-0.03, 1.2-1.4 mm and 0.1-0.4 mm, respectively. Delineations obtained with DeepMedic for a patient in the test set are presented in Fig. The performance of DeepMedic in the preliminary study and after clinical implementation are presented in Table τ= 0.5, 1, 1.5, 2 and 3 mm as reported in Fig. σ) DSCS was 0.98 ±0.03, 0.92 ±0.05, 0.989 ±0.008 and 0.997 ±0.003 for τ=2 mm for bladder, rectum, left and right femur, respectively.Also, the SDSC was calculated for several threshold, The use of MRI for prostate radiotherapy delineation is becoming increasingly common among radiotherapy departments . MRI areIn this study, we demonstrated that deep learning-based approaches can utilise MRI to automatically segment OARs achieving high conformality. Also, a convolutional network has been implemented for clinical use, demonstrating the capability to maintain the performances obtained in a preliminary study.95 and MSD as obtained by Kazemifar et al. and Dong et al. [Table g et al. . Comparig et al. . In thisIn this study, a qualitative assessment by a manual observer has been presented. Unfortunately, it has not been recorded whether the overall time for the delineation has been reduced. Previous studies investigated this aspect  when intThe time necessary for automatic delineation on full FOV is within a minute. Such time-scale can be of interest for conventional radiotherapy and for MR-guided treatments. On the one hand, for conventional radiotherapy, fast automatic OAR segmentation may facilitate the reducing delays in the start of the treatments that may lead to hampered clinical outcomes . On the High conformality for OARs delineation was achieved with two in-house trained networks, obtaining a significant speed-up of the delineation procedure. One of the networks, DeepMedic, was successfully adopted in the clinical workflow maintaining in the clinical setting the accuracy obtained in the feasibility study conducted before clinical implementation.Additional file 1 Configuration files for the network architectures. As part of the supplementary material, it is possible to download the configuration files (ConfigFiles.zip) for DeepMedic  and dV-net (NiftyNet_train.ini). The configuration files for DeepMedic are three and contains the information regarding the model (DeepMedic_model.cfg), training (DeepMedic_train.cfg) and inference (DeepMedic_inference.cfg) of the network.


Recent advances in neurotechnology provide means to non-invasively target these oscillations using frequency-tuned amplitude-modulated transcranial alternating current stimulation (AM-tACS). However, online adaptation of stimulation parameters to ongoing brain oscillations remains an unsolved problem due to stimulation artifacts that impede such adaptation, particularly at the target frequency. Here, we introduce a real-time compatible artifact rejection algorithm  that overcomes this limitation. SASS is a spatial filter (linear projection) removing EEG signal components that are maximally different in the presence versus absence of stimulation. This enables the reliable removal of stimulation-specific signal components, while leaving physiological signal components unaffected. For validation of SASS, we evoked brain activity with known phase and amplitude using 10 Hz visual flickers across 7 healthy human volunteers. 64-channel EEG was recorded during and in absence of 10 Hz AM-tACS targeting the visual cortex. Phase differences between AM-tACS and the visual stimuli were randomized, so that steady-state visually evoked potentials (SSVEPs) were phase-locked to the visual stimuli but not to the AM-tACS signal. For validation, distributions of single-trial amplitude and phase of EEG signals recorded during and in absence of AM-tACS were compared for each participant. When no artifact rejection method was applied, AM-tACS stimulation artifacts impeded assessment of single-trial SSVEP amplitude and phase. Using SASS, amplitude and phase of single trials recorded during and in absence of AM-tACS were comparable. These results indicate that SASS can be used to establish adaptive (closed-loop) AM-tACS, a potentially powerful tool to target various brain functions, and to investigate how AM-tACS interacts with electric brain oscillations. When targeting such frequencies, distinct effects on perception , movemenRecently, we have introduced a novel tACS approach that uses amplitude modulation of a high frequency carrier signal (e.g. 220 Hz) to reduce stimulation-related artifacts at the lower physiological frequency bands . By moduHere, we introduce Stimulation Artifact Source Separation (SASS), a real-time-compatible signal decomposition algorithm, that allows for separating electric brain activity and AM-tACS stimulation artifacts. To test validity and reliability of SASS, brain oscillations with known amplitude and phase were evoked using a 10 Hz steady-state visual evoked potential (SSVEP) paradigm. Brain oscillations were recorded by 64-channel EEG while 10 Hz AM-tACS was applied over the visual cortex. In each trial, phase of AM-tACS relative to the visual flicker was randomly selected, such that the artifact eliminated the consistent phase relationship (phase locking) between the flicker and EEG signal. Using SASS, we successfully recovered single-trial phase and amplitude information of SSVEPs across six healthy human volunteers.22.12.1.1P can be applied to the filtered or unfiltered data. In the present work, we apply P to the filtered data for single-trial SSVEP analysis as well as to broadband data for power spectral analysis. The entire procedure is outlined in SASS is a spatial filter applied to encephalographic data contaminated by transcranial electric stimulation (tES) artifacts. SASS is computed from two covariance matrices, obtained from data bandpass filtered around the stimulation frequency during AM-tACS and in absence of AM-tACS. SASS is computed from and applied to the full-length recordings before any further analysis. It should be noted that, depending on the signal of interest, the SASS projection matrix 2.1.2A, where the resulting components SASS identifies hidden and linearly separable data components that are maximally attributable to transcranial electric stimulation and least attributable to brain activity. These components are then rejected to achieve stimulation artifact suppression. SASS is based on a generalized eigenvalue decomposition  of encephalographic covariance matrices during and in absence of stimulation. It was shown that using an eigenvalue decomposition of the covariance matrix for Signal-Space Projection (SSP)  can be uP can then be constructed, which projects the data onto the vector subspace  orthogonal to the top few components explaining the most variance (“noise space”). This can be written as a separation of the data into latent components, followed by a zeroing out of undesired components and a projection back into the original sensor space:The matrix A) and in absence of stimulation (B), respectively:To account for the spatial distribution of physiological brain activity, SASS was designed to identify components that jointly maximize the variance attributable to stimulation artifacts and minimize the variance attributable to brain activity. In SASS, a source separation matrix is computed from the joint diagonalization of EEG sensor covariance matrices during  with passive Ag/AgCl electrodes  was used to record electrical activity on the scalp. The amplifier was set to DC-mode with a dynamic range of +/-430 V, a resolution of 51 nV/bit, and a range of 24 bit. Electrode impedances were kept below 10 kOhm. Signals were sampled at 500 Hz with an anti-aliasing filter applied at 125 Hz. Saturated electrodes were excluded from the analysis. Electrodes exhibiting broadband power more than two orders of magnitude higher than the median in the condition without AM-tACS were excluded from the analysis.2.3AM-tACS was applied to the scalp using a commercial stimulator . Rubber electrodes with a 4 × 5 cm size were placed over position CPz and on the inion according to the international 10–20 system. This corresponds to the standard montage used for targeting the visual system during tACS experiments e.g., . The sti2.4During ongoing AM-tACS, a sinusoidal grating that flickered at 10 Hz was presented for 2 s across trials. A random inter-trial interval between 0.5 to 1 s ensured that the onset time of the visual flicker was randomly distributed over the phase of the AM-tACS signal. Visual stimuli were presented via a head-mounted display . Its analog audio output was fed into a bipolar channel of the EEG amplifier and stored to obtain a trigger marker of the stimulus onset time. Jitter between the audio output signal and visual stimulus presentation was under 5 ms. Before the experiment, signal artifacts related to the use of the head-mounted display were ruled out.2.5Seven healthy participants  were invited to participate in the study and provided written informed consent. The study was approved by the ethics committee of the Charité – University Medicine Berlin (EA1/077/18). Initially, a calibration session consisting of 200 trials of visual flicker was recorded in absence of AM-tACS. Then, a session of 200 trials of visual flickers was recorded while AM-tACS was applied. Recording time for each participant amounted to approximately 20 min. One participant was excluded due to a lack of discernible SSVEPs in absence of AM-tACS.2.6MNE-Python  was used2.6.1EEG data in absence and during AM-tACS were bandpass filtered around the stimulation frequency from 9–11 Hz using finite impulse response (FIR) filters designed via the Hamming window method . The emp2.6.2To compute power spectra , Welch p2.6.3Following , we asse2.6.4To compute single-trial amplitude and phase, the unsegmented bandpass filtered EEG data was Hilbert transformed. Then, the data was segmented into 2 s trials corresponding to the presentation of each visual flicker. Finally, instantaneous amplitude and phase difference between EEG and flicker was computed for each timepoint and averaged within each trial to obtain a single data point per trial. For visualization, the mean phase angle across trials was subtracted.2.7t-test for independent samples. To test whether single-trial amplitudes during AM-tACS with SASS were smaller than during AM-tACS without SASS, we employed a one-sided t-test for dependent samples. To test whether single-trial amplitudes during AM-tACS with SASS were different from single-trial amplitudes in absence of AM-tACS , we employed a two-sided t-test for independent samples. A power analysis revealed that an effect size of 0.281 (Cohen's d) could be detected with 0.8 power at the employed alpha level of 0.05, meaning that a difference in means  of between 0.174 and 0.972 μV  could be detected.To test whether single-trial amplitudes during AM-tACS without SASS were larger than in absence of AM-tACS, we employed a one-sided Analogously to the case of single-trial amplitudes, we employed Wallraff tests  for depeTo test for differences in single-trial amplitude between conditions across the entire sensor space, spatial cluster-based permutation tests with threshold-free cluster enhancement  were com33.13.23.33.4Depending on the experimental design and purpose of study, there are a number of properties of SASS that users should consider. First, presumably due to spatially varying capacitive effects 4Up to now, there was no real-time compatible signal processing tool available for AM-tACS artifact suppression that allows for recovery of phase and amplitude of evoked brain responses at a single-trial level. Here, we introduced SASS, a spatial filter based on a source separation matrix computed from joint diagonalization of EEG sensor covariance matrixes at the target frequency recorded in absence and during AM-tACS. To evaluate the effectiveness of SASS, we used a 10 Hz-SSVEP paradigm resulting in evoked oscillatory brain responses with known frequency, amplitude and phase. Electric brain activity was assessed using EEG across six healthy volunteers in absence and during AM-tACS targeting the SSVEP frequency. Using SASS, single-trial SSVEP amplitude and phase information was restored to the level recorded in absence of stimulation  across aBesides showing that SASS is an effective tool for separating electric brain activity and stimulation artifacts at the frequency targeted by AM-tACS, our results pave the way for implementation of adaptive stimulation paradigms in which the phase, amplitude and spatial distribution of the applied electric field is adapted to ongoing brain oscillations. This may contribute to the development of more effective stimulation approaches to target various brain functions and further elucidate the underlying mechanisms of AM-tACS.While 200 trials employed in the current study provide sufficient statistical power to detect residual artifacts of above 0.174 and 0.972 μV, respectively,  averaginDespite these considerations, we have shown that SASS allows for successful recovery of single-trial SSVEP amplitude and phase during AM-tACS. Thereby, SASS now allows for further investigation of tACS-related network effects . SASS coReal-time phase estimation of ongoing brain oscillations is challenging and critically depends on the signal-to-noise ratio and instantaneous amplitude of the signal . It is thttp://dx.doi.org/10.17632/fzcmhhjs76.2.The data is publicly available on Mendeley Data: https://github.com/davidhaslacher/sass.The implementation of the novel algorithm  is publicly available on GitHub: D.H., K.N., S.R.S., C.B. and S.E.R. were involved in the design of study, analysis and/or interpretation of data, drafting the manuscript, and revising the manuscript critically for important intellectual content. D.H. and K.N. acquired the data.None.


Hot-deformed anisotropic Nd-Fe-B magnets may potentially attain high coercivity due to their fine and highly orientated crystal grain microstructure as a result of the unique production process that creates these magnets. However, despite their fine grain size of 100–500 nm, coercivity was only around 25% of the full potential of the anisotropy field. This grain size was close to the critical diameter of the single domain grain size of the Nd-Fe-B magnet. This study investigated the effects of chemical composition and deformation conditions on the magnetic properties of Nd-Fe-B magnets, observing their microstructure to obtain guidance on the ideal microstructure. We also improved the hot-deformation technique in parallel to optimize microstructure by controlling the compositions and hot-deformation conditions based on the results of basic studies. Lastly, we fabricated heavy rare-earth-free magnets with a coercivity exceeding 1600 kA/m (20 kOe), which is 20% higher than that of conventional magnets.  In particular, there has been an increasing demand for Nd-Fe-B magnets with high remanence and high heat resistance for HEV traction motors. Generally, these applications require magnets with high remanence, ield, Ha . HoweverTo avoid this resource scarcity issue, there are two ways to improve coercivity with reduced or no HREEs; (1) utilizing the grain boundary diffusion (GBD) process ,6, and  compared to the composition of the R2Fe14B crystal magnetic phase, in order to supply the liquid phase during hot deformation and grain boundary phase for final production. Then, the powder was cold-pressed at room temperature followed by hot-pressing at approximately 800°C to obtain a fully dense body. A hot-pressed body is an isotropic magnet with grains that are approximately 20–50 nm. Finally, to obtain anisotropic magnets, a hot-pressed body was hot-deformed at approximately 800°C; this is the point at which thermo-mechanical alignment of the crystal grains occurs. With the progression of the hot-deformation process, the grain size and its aspect ratio become large . The grains begin to align gradually, at the same time and the direction of the c-axis (easy axis) is perpendicular to the compression direction. The hot-pressing and hot-deformation temperatures were kept higher than the melting point of the grain boundary phase to ensure good formability whilst pressing. However, one of the reasons for abnormal grain growth was excess heat input during hot-pressing and hot-deformation [ormation ,13. ThusIn 2.2.Hot-deformation is usually completed in several tens of seconds, which means that significant microstructural change is completed within a short period of time. Specific mechanisms have been proposed to explain this phenomenon, although a consensus is still lacking on whether these mechanisms are indeed the cause of this phenomenon. In this section, we introduce our research group’s results and propose possible mechanisms from our results  and prevR, is defined by the equation below, where h0 and h are the height before and after die-upsetting:The schematic models of possible mechanisms our research group supports are described in a-axis of the R2Fe14B crystal  [2Fe14B crystal was in the a-axis, as reported in previous studies [R became larger. The grain size and its aspect ratio simultaneously became larger, whilst no dislocations or slip lines were observed in normal area [ragonal) . Based o studies . In Figumal area .c-axis of platelet grains align parallel to the press direction. It is conceivable the anisotropic grain growth during hot deformation is mainly caused by the atom transfer through the grain boundary phase because chemical composition of grain boundary phase changes during the microstructure evolution [Therefore, we inferred possible mechanism as follows. First, the grain boundary phase melts and anisotropic grain growth occurs during hot pressing. Then, the anisotropic grain growth and crystal rotation with the grain boundary sliding of the c-plane (broad surface of platelet-shaped grain) are promoted by a stress under hot deforming . Here, tvolution . Howevervolution ,13,17 anvolution ,20 have 2Fe14B crystal was also proposed. In this mechanism, Young’s modulus of the c-axis is lower than the a-axis [a-axis under compression [Another possible mechanism based on the elastic anisotropy of the Re a-axis ; as suchpression ,23 and sBased on the previous studies, it is apparent that one specific mechanism cannot completely account for the entire orientation phenomenon, although these mechanisms  are likely to be intricately related.3.3.1.c-axis, respectively. As shown in c-axis direction; this is one order of magnitude finer than the sintered magnet. The grain size was similar to the critical diameter of single-domain Nd2Fe14B grains (0.3 μm) [Microstructure observations were conducted to understand the features of hot-deformed Nd-Fe-B magnets. (0.3 μm) .Table 1.3.2.3.3.In 4.4.1.The effects of chemical composition and microstructure , on the magnetic properties of hot-deformed Nd-Fe-B magnets were systematically evaluated . Table 2J1 is the inflection point of initial magnetization curves and JS is the saturation magnetization.2Fe14B grains. The SDGRs of all samples A–E increase as the average grain size decreases, which indicates the increasing volume of single-domain structure corresponds to decreasing average grain size.c-axis. As seen in the figure, it is clear that the crystal grains grow as the hot-deformation temperature increases.β and α, between room temperature and 180°C for all samples, where α and β are given by:α values of all samples were around −0.1%/°C; they are unaffected by either grain size or the TRE. On the other hand, the β values of all samples improved with the addition of Dy, the increased TRE content, and the grain size reduction. This result shows that it is possible to obtain high heat resistance without HREEs by reducing grain size. Besides, it was found that the increasing volume of single-domain structure affects not only coercivity but also improvement of the β values from From Hcj = 720 (9.0 kOe), 1030 (12.9 kOe), and 1430 kA/m (17.9 kOe), respectively. The coercivity increased with the Nd amount, as observed for Samples A–C. They observed the thickness and chemical composition of the grain boundary phase using TEM, LV-SEM, and three-dimensional atomic probe (3DAP). The observations showed that the thickness of the grain boundary phases increased with the Nd content. In this study, the average Nd content in the grain boundary phase increased from 22.7.% to 46.0 at.% with an increase in thickness. These results suggest that the increase of Nd at the grain boundary phase enhances the magnetic isolation of the grains.Liu et al.  made micAnother microstructural analysis of the effect of grain size was also reported. Sepheri-Amin et al.  observedThese studies found that there were improvements to the heat resistance of hot-deformed magnets through the control of the microstructure without the addition of HREEs. Some simulations also support these experimental results .4.2.c-axis. The sample was first fully magnetized, such that almost all grains were magnetized in the same direction at 30°C  and lengths (maximum 80 mm) may be produced;The magnetic flux is uniform along the height direction even if the ring magnet has a great height. This is because it does not require grain alignment under a magnetic field;Various magnetization patterns and precise magnetization waveform control were possible by controlling mold structure;Relatively good corrosion resistance; and Near net-shape manufacturing was possible.6.To improve the accuracy in controlling the microstructure, further investigation of the orientation mechanism as described in To provide magnets for newly designed motors , hot-defApplication of grain boundary engineering techniques such as the GBD process to hot-deformed magnets for a remarkable increase in coercivity with small HREE addition ;Recently, it has been possible to experimentally observe the magnetization reversal of a few particles . From suTo avoid resource problems, low-cost REEs such as Ce and La need to be used efficiently. It is clear that the magnetic properties become worse by substituting them with Nd. However, there are reports that the magnetic properties do not worse as much as the physical properties by optimizing composition and microstructure ,41. For Increase the electrical resistivity of the magnet material. During motor operation, the temperature of the magnets increases due to the eddy current at the surface of the magnets. The higher electrical resistivity of magnets is effective for reducing eddy currents. For hot-deformed magnets, there are studies investigating into mixing high-electrical resistivity material with raw powder, followed by fabricating magnets ,45. HoweIn order to improve the coercivity of hot-deformed Nd-Fe-B magnets without HREEs addition, we investigated the effects of chemical composition and hot deformation conditions on the magnetic properties of the magnets, observing their microstructures to obtain guidance on the ideal microstructure. Based on these results, we optimized compositions and hot-deformation conditions to enhance the coercivity by refining grains and promoting grain isolation magnetically and to improve the remanence by eliminating abnormally growth grain areas. Consequently, we successfully developed high performance hot-deformed magnets that had greater than 1600 kA/m (20 kOe) of coercivity without any addition of HREEs, which are used for HEV traction motors . However


Escherichia coli-based anode-assisted fermentation of glucose to acetoin. The experiments build on an obligate respiratory strain, that was advanced using selective adaptation and targeted strain development. Several transfers under respiratory conditions led to point mutations in the pfl, aceF and rpoC gene. These mutations increased anoxic growth by three-fold. Furthermore, overexpression of genes encoding a synthetic electron transport chain to methylene blue increased the electron transfer rate by 2.45-fold. Overall, these measures and a medium optimization increased the space-time yield in an electrode-assisted fermentation by 3.6-fold.Anode-assisted fermentations offer the benefit of an anoxic fermentation routine that can be applied to produce end-products with an oxidation state independent from the substrate. The whole cell biocatalyst transfers the surplus of electrons to an electrode that can be used as a non-depletable electron acceptor. So far, anode-assisted fermentations were shown to provide high carbon efficiencies but low space-time yields. This study aimed at increasing space-time yields of an  ΔatpFH) . Th. ThpflB ocalized ,30. Althocalized . Still, ocalized ,32,33,34ositions . Neverthoacetate . These teactions ,38. As thenotype . Neverthhenotype . Overall600 = 6. The chronoamperometric measurement of the electro-fermentation at OD600 = 6 revealed an initial current density maximum of 667.7 ± 83.6 mA/m2 after 4 min followed by a slow decrease in current density . The plasmid also contained the alsSD genes for acetoin production. The introduction of the plasmid and expression of the genes increased the methylene blue reduction rate by 2.45-fold  and consequently the productivity (3.6-fold) that can be achieved with this strain. KM value of 273 µmol/L was established for the whole-cell biocatalyst. Using this data and converting the measured reduction rates to a potentially achievable current density leads to a value of approximately 1.55 A/m2. While the initial current densities observed in the electro-fermentation experiment are only 2.3-fold lower as this value, the average current density throughout 8 h is 4.8-fold lower. This has the following implications: First, the development of the strain was successful as it is easily imaginable that the initially discussed mass transfer limitations of the BES cause the 2.3-fold difference in electron transfer rate. Second, this difference becomes more pronounced over longer periods is most probably due to the growth-inhibiting effect of methylene blue that is not interfering with the fast kinetics measured in cell suspension experiments. Hence, a further step towards a more stable current production would be to establish an E. coli strain that is more resilient towards higher methylene blue concentrations. Previous studies already revealed potential targets for a directed strain evolution approach. Apparently, the efflux pump AcrAB is necessary for E. coli to thrive in medium with higher methylene blue concentrations [This study started with establishing the characteristics of methylene blue reduction kinetics and a trations ,44. A deIncreasing space-time yields of electrode-assisted fermentations will be key for their future biotechnological implementation. The here conducted study provides a workflow for strain development and characterization of the capabilities of the applied BES. Further research will focus on accelerating glucose metabolism in the strain and aim at increasing the amount of active biocatalyst within the bioreactors by advancing reactor technology.